4&#39;&#39;-Substituted Erythromycin Derivative

ABSTRACT

A compound of formula (I) 
     
       
         
         
             
             
         
       
     
     and/or pharmaceutically acceptable derivatives thereof, having antimicrobial activity, processes for their preparation, compositions containing them and to their use in medicine.

The present invention relates to novel semi-synthetic macrolides havingantimicrobial activity, in particular antibacterial activity. Moreparticularly, the invention relates to 14-membered macrolidessubstituted at the 4″ position, processes for their preparation,compositions containing them and to their use in medicine.

Macrolide antibacterial agents are known to be useful in the treatmentor prevention of bacterial infections. However, the emergence ofmacrolide-resistant bacterial strains has resulted in the need todevelop new macrolide compounds. For example, EP 0 895 999, WO03/042228, WO 04/101585, WO 04/101586, WO 04/101587, WO 04/101588, WO04/101589, WO 04/101590, WO 04/039822, WO 05/108412, and WO 05/108413describe derivatives modified at the 4″ position of the macrolide havingantibacterial activity.

According to the present invention, we have now found novel 14-memberedmacrolides substituted at the 4″ position which also have antimicrobialactivity.

Thus, the present invention provides the compound of formula (I)

and/or pharmaceutically acceptable derivatives thereof.

The term “pharmaceutically acceptable” as used herein means a compoundwhich is suitable for pharmaceutical use. Salts and solvates ofcompounds of the invention which are suitable for use in medicine arethose wherein the counterion or associated solvent is pharmaceuticallyacceptable. However, salts and solvates having non-pharmaceuticallyacceptable counterions or associated solvents are within the scope ofthe present invention, for example, for use as intermediates in thepreparation of other compounds of the invention and theirpharmaceutically acceptable salts and solvates.

The term “derivative” as used herein means any salt, solvate or prodrug,e.g. ester, of the compound of the invention, which upon administrationto the recipient is capable of providing (directly or indirectly) acompound of the invention, or an active metabolite or residue thereof.Such derivatives are recognizable to those skilled in the art, withoutundue experimentation.

The term “pharmaceutically acceptable derivative” as used herein meansany pharmaceutically acceptable salt, solvate or prodrug, e.g. ester, ofthe compound of the invention, which upon administration to therecipient is capable of providing (directly or indirectly) a compound ofthe invention, or an active metabolite or residue thereof. Suchderivatives are recognizable to those skilled in the art, without undueexperimentation. Nevertheless, reference is made to the teaching ofBurger's Medicinal Chemistry and Drug Discovery, 5^(th) Edition, Vol 1:Principles and Practice, which is incorporated herein by reference tothe extent of teaching such derivatives. Examples of pharmaceuticallyacceptable derivatives are salts, solvates, esters, carbamates andphosphate esters. Additional examples of pharmaceutically acceptablederivatives are salts, solvates and esters. Further examples ofpharmaceutically acceptable derivatives are salts and esters, such assalts.

The term “alkyl” as used herein as a group or a part of a group refersto a straight or branched hydrocarbon chain. Examples of “alkyl” as usedherein include, but are not limited to, methyl, ethyl, n-propyl,n-butyl, n-pentyl, isobutyl, isopropyl, t-butyl, hexyl. In one aspect,“alkyl” refers to a C₁₋₄alkyl group, for example methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl or t-butyl.

The compound of the present invention may be in the form of and/or maybe administered as a pharmaceutically acceptable salt. For a review onsuitable salts see Berge et al., J. Pharm. Sci., 1977, 66, 1-19.

Typically, a pharmaceutically acceptable salt may be readily prepared byusing a desired acid or base as appropriate. The salt may precipitatefrom solution and be collected by filtration or may be recovered byevaporation of the solvent. For example, an aqueous solution of an acidsuch as lactobionic acid may be added to a solution of a compound offormula (I) in a solvent such as acetonitrile, acetone or THF, and theresulting mixture evaporated to dryness, redissolved in water andlyophilised to obtain the acid addition salt as a solid. Alternatively,a compound of formula (I) may be dissolved in a suitable solvent, forexample an alcohol such as isopropanol, and the acid may be added in thesame solvent or another suitable solvent. The resulting acid additionsalt may then be precipitated directly, or by addition of a less polarsolvent such as diisopropyl ether or hexane, and isolated by filtration.

The skilled person will appreciate that the compound of formula (I)contains more than one basic group and therefore either mono (1:1acid:compound of formula (I)) or bis salts (2:1) may be formed and aresalts according to the present invention.

Suitable addition salts are formed from inorganic or organic acids whichform non-toxic salts and examples are lactobionate, mandelate (including(S)-(+)-mandelate, (R)-(−)-mandelate and (R,S)-mandelate),hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate,phosphate, hydrogen phosphate, acetate, trifluoroacetate, maleate,malate, fumarate, lactate, tartrate, citrate, formate, gluconate,succinate, ethyl succinate (4-ethoxy-4-oxo-butanoate), pyruvate,oxalate, oxaloacetate, saccharate, benzoate, sulphonates (e.g.methanesulphonate, ethanesulphonate, benzenesulphonate orp-toluenesulphonate) and isethionate. In one embodiment, suitable saltsinclude phosphate, fumarate, and tartrate, for example bis-phosphate,D-tartrate and bis-fumarate.

Pharmaceutically acceptable base salts include ammonium salts, alkalimetal salts such as those of sodium and potassium, alkaline earth metalsalts such as those of calcium and magnesium and salts with organicbases, including salts of primary, secondary and tertiary amines, suchas isopropylamine, diethylamine, ethanolamine, trimethylamine,dicyclohexyl amine and N-methyl-D-glucamine.

The compound of the invention has both a basic and an acidic centre maytherefore be in the form of a zwitterion.

Those skilled in the art of organic chemistry will appreciate that manyorganic compounds can form complexes with solvents in which they arereacted or from which they are precipitated or crystallized. Thesecomplexes are known as “solvates”. For example, a complex with water isknown as a “hydrate”. Solvates of the compounds of the invention arewithin the scope of the invention. The salts of the compound of formula(I) may form solvates (e.g. hydrates) and the invention also includesall such solvates.

The term “prodrug” as used herein means a compound which is convertedwithin the body, e.g. by hydrolysis in the blood, into its active formthat has medical effects. Pharmaceutically acceptable prodrugs aredescribed in T. Higuchi and V. Stella, “Prodrugs as Novel DeliverySystems”, Vol. 14 of the A.C.S. Symposium Series; Edward B. Roche, ed.,“Bioreversible Carriers in Drug Design”, American PharmaceuticalAssociation and Pergamon Press, 1987; and in D. Fleisher, S. Ramon andH. Barbra “Improved oral drug delivery: solubility limitations overcomeby the use of prodrugs”, Advanced Drug Delivery Reviews (1996) 19(2)115-130, each of which are incorporated herein by reference.

Prodrugs are any covalently bonded carriers that release a compound offormula (I) in vivo when such prodrug is administered to a patient.Prodrugs are generally prepared by modifying functional groups in a waysuch that the modification is cleaved, either by routine manipulation orin vivo, yielding the parent compound. Prodrugs include, for example,compounds of this invention wherein hydroxy, amine or sulfhydryl groupsare bonded to any group that, when administered to a patient, cleaves toform the hydroxy, amine or sulfhydryl groups. Thus, representativeexamples of prodrugs include (but are not limited to) acetate, formateand benzoate derivatives of alcohol, sulfhydryl and amine functionalgroups of the compounds of formula (I). Further, in the case of acarboxylic acid (—COOH), esters may be employed, such as methyl esters,ethyl esters, and the like. Esters may be active in their own rightand/or be hydrolysable under in vivo conditions in the human body.Suitable pharmaceutically acceptable in vivo hydrolysable ester groupsinclude those which break down readily in the human body to leave theparent acid or its salt.

References hereinafter to a compound according to the invention includeboth compounds of formula (I) and their pharmaceutically acceptablederivatives.

With regard to stereoisomers, the compound of formula (I) has more thanone asymmetric carbon atom. In the general formula (I) as drawn, thesolid wedge shaped bond indicates that the bond is above the plane ofthe paper. The broken wedge shaped bond indicates that the bond is belowthe plane of the paper.

It will be appreciated that the substituents on the macrolide may alsohave one or more asymmetric carbon atoms, for example in a prodrug estergroup. Thus, the compound of formula (I) may occur as individualenantiomers or diastereomers. All such isomeric forms are includedwithin the present invention, including mixtures thereof.

Separation of diastereoisomers or cis and trans isomers may be achievedby conventional techniques, e.g. by fractional crystallisation,chromatography or HPLC. A stereoisomeric mixture of the agent may alsobe prepared from a corresponding optically pure intermediate or byresolution, such as by HPLC, of the corresponding mixture using asuitable chiral support or by fractional crystallisation of thediastereoisomeric salts formed by reaction of the corresponding mixturewith a suitable optically active acid or base, as appropriate.

The compounds of the invention may be in crystalline or amorphous form.Furthermore, some of the crystalline forms of the compounds of theinvention may exist as polymorphs, which are included in the presentinvention.

It is to be understood that the present invention covers allcombinations of the embodiments and representative examples describedhereinabove.

Compounds of the invention include:

-   4″-O-{2-[(3-[3-Carboxy-1-dimethylamino-4-oxo-1,4-dihydro-6-quinolinyl]propyl)methylamino]ethyl}-6-O-methyl-erythromycin    A    or pharmaceutically acceptable derivatives thereof.

Compounds according to the invention may exhibit a broad spectrum ofantimicrobial activity, in particular antibacterial activity, against awide range of clinical pathogenic microorganisms. Using a standardmicrotiter broth serial dilution test, compounds of the invention havebeen found to exhibit useful levels of activity against one or more of arange of pathogenic microorganisims, for example gram positive bacteria.The compounds of the invention may be active against strains whichinclude Staphylococcus aureus, Streptopococcus pneumoniae, Moraxellacatarrhalis, Streptococcus pyogenes, Haemophilus influenzae,Enterococcus faecalis, Chlamydia pneumoniae, Mycoplasma pneumoniae andLegionella pneumophila, such as Staphylococcus aureus, Streptopococcuspneumoniae, Enterococcus faecalis and Streptococcus pyogenes. Thecompounds of the invention may also be active against resistant strains,for example erythromycin resistant strains. Thus, for example, thecompounds of the invention may be active against erythromycin resistantstrains of Streptococcus pneumoniae, Streptococcus pyogenes andStaphylococcus aureus.

The compounds of the invention may therefore be useful for treating avariety of diseases caused by pathogenic microorganisms, in particularbacteria, in human beings and animals. It will be appreciated thatreference to treatment includes acute treatment or prophylaxis as wellas the alleviation of established symptoms.

The compounds of the invention may also be more efficacious, showgreater selectivity, have fewer side effects, have a longer duration ofaction, be more bioavailable by the preferred administration route, havemore suitable pharmacodynamic or pharmacokinetic properties, or haveother more desirable properties, for example, have better physicalproperties such as crystallinity than similar known compounds.

Thus, according to another aspect of the present invention we provide acompound of formula (I) or a pharmaceutically acceptable derivativethereof for use in therapy.

According to a further aspect of the invention we provide a compound offormula (I) or a pharmaceutically acceptable derivative thereof for usein the treatment or prophylaxis of systemic or topical microbialinfections in a human or animal body.

According to a further aspect of the invention we provide the use of acompound of formula (I) or a pharmaceutically acceptable derivativethereof in the manufacture of a medicament for use in the treatment orprophylaxis of systemic or topical microbial infections in a human oranimal body.

According to a yet further aspect of the invention we provide a methodof treatment of the human or non-human animal body to combat microbialinfections comprising administration to a body in need of such treatmentof an effective amount of a compound of formula (I) or apharmaceutically acceptable derivative thereof.

Examples of disease states in which the compounds of the invention mayhave utility include skin diseases such as eczema, psoriasis, allergicdermatitis, neurodermatitis, pruritis and hypersensitivity reactions;inflammatory conditions of the nose, throat or lungs such as asthma(including allergen-induced asthmatic reactions), rhinitis (includinghayfever), nasal polyps, chronic obstructive pulmonary disease,interstitial lung disease, and fibrosis; inflammatory bowel conditionssuch as ulcerative colitis and Crohn's disease; atherosclerosis;diabetes after ischemia and reperfusion; myocardial infarction; stroke;cirrhosis; and auto-immune diseases such as rheumatoid arthritis.

Compounds of the invention may also have use in the treatment ofconjunctiva and conjunctivitis.

While it is possible that, for use in therapy, a compound of theinvention may be administered as the raw chemical it is preferable topresent the active ingredient as a pharmaceutical formulation e.g. whenthe agent is in admixture with a suitable pharmaceutical excipient,diluent or carrier selected with regard to the intended route ofadministration and standard pharmaceutical practice.

Accordingly, in one aspect, the present invention provides apharmaceutical composition or formulation comprising a compound of theinvention or a pharmaceutically acceptable derivative thereof inassociation with a pharmaceutically acceptable excipient, diluent and/orcarrier. The excipient, diluent and/or carrier must be “acceptable” inthe sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

In another aspect, the invention provides a pharmaceutical compositioncomprising, as active ingredient, a compound of the invention or apharmaceutically acceptable derivative thereof in association with apharmaceutically acceptable excipient, diluent and/or carrier for use intherapy, and in particular, in the treatment of human or animal subjectssuffering from a condition susceptible to amelioration by anantimicrobial compound.

In another aspect, the invention provides a pharmaceutical compositioncomprising a therapeutically effective amount of a compound of thepresent invention and a pharmaceutically acceptable excipient, diluentand/or carrier (including combinations thereof).

There is further provided by the present invention a process ofpreparing a pharmaceutical composition, which process comprises mixing acompound of the invention or a pharmaceutically acceptable derivativethereof, together with a pharmaceutically acceptable excipient, diluentand/or carrier.

The compounds of the invention may be formulated for administration inany convenient way for use in human or veterinary medicine and theinvention therefore includes within its scope pharmaceuticalcompositions comprising a compound of the invention adapted for use inhuman or veterinary medicine. Such compositions may be presented for usein a conventional manner with the aid of one or more suitableexcipients, diluents and/or carriers. Acceptable excipients, diluentsand carriers for therapeutic use are well known in the pharmaceuticalart, and are described, for example, in Remington's PharmaceuticalSciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice ofpharmaceutical excipient, diluent and/or carrier can be selected withregard to the intended route of administration and standardpharmaceutical practice. The pharmaceutical compositions may compriseas—or in addition to—the excipient, diluent and/or carrier any suitablebinder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s).

Preservatives, stabilisers, dyes and even flavouring agents may beprovided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

For some embodiments, the agents of the present invention may also beused in combination with a cyclodextrin. Cyclodextrins are known to forminclusion and non-inclusion complexes with drug molecules. Formation ofa drug-cyclodextrin complex may modify the solubility, dissolution rate,bioavailability and/or stability property of a drug molecule.Drug-cyclodextrin complexes are generally useful for most dosage formsand administration routes. As an alternative to direct complexation withthe drug the cyclodextrin may be used as an auxiliary additive, e.g. asa carrier, diluent or solubiliser. Alpha-, beta- and gamma-cyclodextrinsare most commonly used and suitable examples are described in WO91/11172, WO 94/02518 and WO 98/55148.

The compounds of the invention may be milled using known millingprocedures such as wet milling to obtain a particle size appropriate fortablet formation and for other formulation types. Finely divided(nanoparticulate) preparations of the compounds of the invention may beprepared by processes known in the art, for example see InternationalPatent Application No. WO 02/00196 (SmithKline Beecham).

The routes for administration (delivery) include, but are not limitedto, one or more of: oral (e.g. as a tablet, capsule, or as an ingestablesolution), topical, mucosal (e.g. as a nasal spray or aerosol forinhalation), nasal, parenteral (e.g. by an injectable form),gastrointestinal, intraspinal, intraperitoneal, intramuscular,intravenous, intrauterine, intraocular, intradermal, intracranial,intratracheal, intravaginal, intracerebroventricular, intracerebral,subcutaneous, ophthalmic (including intravitreal or intracameral),transdermal, rectal, buccal, epidural and sublingual.

There may be different composition/formulation requirements depending onthe different delivery systems. By way of example, the pharmaceuticalcomposition of the present invention may be formulated to be deliveredusing a mini-pump or by a mucosal route, for example, as a nasal sprayor aerosol for inhalation or ingestable solution, or parenterally inwhich the composition is formulated by an injectable form, for delivery,by, for example, an intravenous, intramuscular or subcutaneous route.Alternatively, the formulation may be designed to be delivered by bothroutes.

Where the agent is to be delivered mucosally through thegastrointestinal mucosa, it should be able to remain stable duringtransit though the gastrointestinal tract; for example, it should beresistant to proteolytic degradation, stable at acid pH and resistant tothe detergent effects of bile.

Where appropriate, the pharmaceutical compositions can be administeredby inhalation, in the form of a suppository or pessary, topically in theform of a lotion, solution, cream, ointment or dusting powder, by use ofa skin patch, orally in the form of tablets containing excipients suchas starch or lactose, or in capsules or ovules either alone or inadmixture with excipients, or in the form of elixirs, solutions orsuspensions containing flavouring or colouring agents, or they can beinjected parenterally, for example intravenously, intramuscularly orsubcutaneously. For parenteral administration, the compositions may bebest used in the form of a sterile aqueous solution which may containother substances, for example enough salts or monosaccharides to makethe solution isotonic with blood. For buccal or sublingualadministration the compositions may be administered in the form oftablets or lozenges which can be formulated in a conventional manner.

It is to be understood that not all of the compounds need beadministered by the same route. Likewise, if the composition comprisesmore than one active component, then those components may beadministered by different routes.

The compositions of the invention include those in a form especiallyformulated for parenteral, oral, buccal, rectal, topical, implant,ophthalmic, nasal or genito-urinary use. For some applications, theagents of the present invention are delivered systemically (such asorally, buccally, sublingually), more preferably orally. Hence,preferably the agent is in a form that is suitable for oral delivery.

If the compound of the present invention is administered parenterally,then examples of such administration include one or more of:intravenously, intraarterially, intraperitoneally, intrathecally,intraventricularly, intraurethrally, intrasternally, intracranially,intramuscularly or subcutaneously administering the agent, and/or byusing infusion techniques.

For parenteral administration, the compound is best used in the form ofa sterile aqueous solution which may contain other substances, forexample, enough salts or glucose to make the solution isotonic withblood. The aqueous solutions should be suitably buffered (preferably toa pH of from 3 to 9), if necessary. The preparation of suitableparenteral formulations under sterile conditions is readily accomplishedby standard pharmaceutical techniques well-known to those skilled in theart.

The compounds according to the invention may be formulated for use inhuman or veterinary medicine by injection (e.g. by intravenous bolusinjection or infusion or via intramuscular, subcutaneous or intrathecalroutes) and may be presented in unit dose form, in ampoules, or otherunit-dose containers, or in multi-dose containers, if necessary with anadded preservative. The compositions for injection may be in the form ofsuspensions, solutions, or emulsions, in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilising,solubilising and/or dispersing agents. Alternatively the activeingredient may be in sterile powder form for reconstitution with asuitable vehicle, e.g. sterile, pyrogen-free water, before use.

The compounds of the invention can be administered (e.g. orally ortopically) in the form of tablets, capsules, ovules, elixirs, solutionsor suspensions, which may contain flavouring or colouring agents, forimmediate-, delayed-, modified-, sustained-, pulsed- orcontrolled-release applications.

The compounds of the invention may also be presented for human orveterinary use in a form suitable for oral or buccal administration, forexample in the form of solutions, gels, syrups, mouth washes orsuspensions, or a dry powder for constitution with water or othersuitable vehicle before use, optionally with flavouring and colouringagents. Solid compositions such as tablets, capsules, lozenges,pastilles, pills, boluses, powder, pastes, granules, bullets or premixpreparations may also be used. Solid and liquid compositions for oraluse may be prepared according to methods well known in the art. Suchcompositions may also contain one or more pharmaceutically acceptablecarriers and excipients which may be in solid or liquid form.

The tablets may contain excipients such as microcrystalline cellulose,lactose, sodium citrate, calcium carbonate, calcium sulphate, dibasiccalcium phosphate and glycine, manitol, pregelatinised starch, cornstarch, potato starch, disintegrants such as sodium starch glycollate,croscarmellose sodium and certain complex silicates, and granulationbinders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose(HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia.

Additionally, lubricating agents such as magnesium stearate, stearicacid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers ingelatin or HPMC (hydroxypropyl methylcellulose) capsules. Preferredexcipients in this regard include microcrystalline cellulose, lactose,calcium carbonate, calcium sulphate, dibasic calcium phosphate and,manitol, pregelatinised starch, corn starch, potato starch or highmolecular weight polyethylene glycols. For aqueous suspensions and/orelixirs, the agent may be combined with various sweetening or flavouringagents, colouring matter or dyes, with emulsifying and/or suspendingagents and with diluents such as water, ethanol, propylene glycol andglycerin, and combinations thereof.

Capsules, may be filled with a powder (of medicament alone or as blendwith selected filler(s)) or alternatively a liquid, each comprising oneor more compounds of formula (I) and a carrier. Where the capsule isfilled with a powder the compounds of formula (I) and/or the carrier maybe milled or micronised to provide material with an appropriate particlesize.

Compounds of the invention may be susceptible to acid degradation afteringestion and may therefore require a coating, such as an entericcoating, when administered orally as a tablet or capsule.

The tablet or capsule, as appropriate, may, for example be coated by athin film such as a EUDRAGIT® film available from Röhm Pharma Polymers,which allows controlled dissolution in the gastrointestinal tract. Thefilms are available as cationic polymers such as EUDRAGIT® E 100(aminoalkyl methacylate copolymers) or as anionic acrylic polymers suchas EUDRAGIT® L (methacrylic acid copolymers) and EUDRAGIT S. Permeableacrylic polymers such as EUDRAGIT® RL (ammonio methacrylate copolymer)and EUDRAGIT® RS are also available.

These coating formulations may be prepared as an aqueous dispersionincluding optional ingredients such as talc, silicone antifoam emulsion,polyethylene glycol. Alternatively the coating formulation may beprepared as an organic polymer solution.

Alternatively, tablets may be coated using OPADRY® (Surelease®) coatingsystems, available from Colorcon. Aqueous systems generally comprise upto 15% w/w of OPADRY®. Organic solvent systems generally comprise up to5% w/w of OPADRY®.

The coatings may be prepared by known techniques, for example by;

-   -   1. weighing the required quantity of OPADRY® film coating        system,    -   2. weighing the required quantity of water or other solvent(s)        into a mixing vessel,    -   3. with a mixing propeller in the centre of the vessel and as        close to the bottom of the vessel as possible, stirring the        solvents to form a vortex without drawing air into the liquid,    -   4. steadily and quickly adding the OPADRY® powder to the vortex,        avoiding powder flotation on the liquid surface,    -   5. increasing the stirrer speed in order to maintain the vortex,        if required, and    -   6. after all the powder ingredients have been added, reducing        the mixer speed and continuing mixing for approximately 45        minutes.

Coatings can be applied by known techniques, using tablet coatingmachines.

The thickness of the coating applied is generally in the range 5 to 35microns such as 10 to 30 microns, more specifically 10 or 20 microns,depending on the required effect.

Alternatively, the tablet or a capsule, as appropriate, may be filledinto another capsule (preferably a HPMC capsule such as Capsugel®) toprovide either a tablet in capsule or capsule in capsule configuration,which when administered to a patient yields controlled dissolution inthe gastrointestinal tract thereby providing a similar effect to anenteric coating.

Thus in one aspect the invention provides a solid dose formulation of acompound of formula (I) wherein said formulation has an enteric coating.

In another aspect the invention provides a solid dose formulationcomprising a protective capsules as outer layer, for example as a tabletin a capsule or as a capsule in a capsule.

The compounds of the invention may also be administered orally inveterinary medicine in the form of a liquid drench such as a solution,suspension or dispersion of the active ingredient together with apharmaceutically acceptable carrier or excipient.

The compounds of the invention may also, for example, be formulated assuppositories e.g. containing conventional suppository bases for use inhuman or veterinary medicine or as pessaries e.g. containingconventional pessary bases.

The compounds according to the invention may be formulated for topicaladministration, for use in human and veterinary medicine, in the form ofointments, creams, gels, hydrogels, lotions, solutions, shampoos,powders (including spray or dusting powders), pessaries, tampons,sprays, dips, aerosols, drops (e.g. eye ear or nose drops) or pour-ons.

For application topically to the skin, the agent of the presentinvention can be formulated as a suitable ointment containing the activecompound suspended or dissolved in, for example, a mixture with one ormore of the following: mineral oil, liquid petrolatum, white petrolatum,propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifyingwax and water.

Alternatively, it can be formulated as a suitable lotion or cream,suspended or dissolved in, for example, a mixture of one or more of thefollowing: mineral oil, sorbitan monostearate, a polyethylene glycol,liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

The compounds may also be dermally or transdermally administered, forexample, by use of a skin patch.

For ophthalmic use, the compounds can be formulated as micronisedsuspensions in isotonic, pH adjusted, sterile saline, or, preferably, assolutions in isotonic, pH adjusted, sterile saline, optionally incombination with a preservative such as a benzylalkonium chloride.Alternatively, they may be formulated in an ointment such as petrolatum.

As indicated, the compound of the present invention can be administeredintranasally or by inhalation and is conveniently delivered in the formof a dry powder inhaler or an aerosol spray presentation from apressurised container, pump, spray or nebuliser with the use of asuitable propellant, e.g. a hydrofluoroalkane such as1,1,1,2-tetrafluoroethane (HFA 134AT″″) or1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA), carbon dioxide or othersuitable gas. In the case of a pressurised aerosol, the dosage unit maybe determined by providing a valve to deliver a metered amount. Thepressurised container, pump, spray or nebuliser may contain a solutionor suspension of the active compound, e.g. using a mixture of ethanoland the propellant as the solvent, which may additionally contain alubricant, e.g. sorbitan trioleate.

Capsules and cartridges (made, for example, from gelatin) for use in aninhaler or insufflator may be formulated to contain a powder mix of thecompound and a suitable powder base such as lactose or starch.

For topical administration by inhalation the compounds according to theinvention may be delivered for use in human or veterinary medicine via anebuliser.

The compounds of the invention may also be used in combination withother therapeutic agents. The invention thus provides, in a furtheraspect, a combination comprising a compound of the invention or apharmaceutically acceptable derivative thereof together with a furthertherapeutic agent.

When a compound of the invention or a pharmaceutically acceptablederivative thereof is used in combination with a second therapeuticagent active against the same disease state the dose of each compoundmay differ from that when the compound is used alone. Appropriate doseswill be readily appreciated by those skilled in the art. It will beappreciated that the amount of a compound of the invention required foruse in treatment will vary with the nature of the condition beingtreated and the age and the condition of the patient and will beultimately at the discretion of the attendant physician or veterinarian.The compounds of the present invention may for example be used fortopical administration with other active ingredients such ascorticosteroids or antifungals as appropriate.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical formulation and thus pharmaceuticalformulations comprising a combination as defined above together with apharmaceutically acceptable carrier or excipient comprise a furtheraspect of the invention. The individual components of such combinationsmay be administered either sequentially or simultaneously in separate orcombined pharmaceutical formulations by any convenient route.

When administration is sequential, either the compound of the inventionor the second therapeutic agent may be administered first. Whenadministration is simultaneous, the combination may be administeredeither in the same or different pharmaceutical composition.

When combined in the same formulation it will be appreciated that thetwo compounds must be stable and compatible with each other and theother components of the formulation. When formulated separately they maybe provided in any convenient formulation, conveniently in such manneras are known for such compounds in the art.

The compositions may contain from 0.01-99% of the active material. Fortopical administration, for example, the composition will generallycontain from 0.01-10%, more preferably 0.01-1% of the active material.

Typically, a physician will determine the actual dosage which will bemost suitable for an individual subject. The specific dose level andfrequency of dosage for any particular individual may be varied and willdepend upon a variety of factors including the activity of the specificcompound employed, the metabolic stability and length of action of thatcompound, the age, body weight, general health, sex, diet, mode and timeof administration, rate of excretion, drug combination, the severity ofthe particular condition, and the individual undergoing therapy.

For oral and parenteral administration to humans, the daily dosage levelof the agent may be in single or divided doses.

For systemic administration the daily dose as employed for adult humantreatment it will range from 2-100 mg/kg body weight, preferably 5-60mg/kg body weight, which may be administered in 1 to 4 daily doses, forexample, depending on the route of administration and the condition ofthe patient. When the composition comprises dosage units, each unit willpreferably contain 200 mg to 1 g of active ingredient. The duration oftreatment will be dictated by the rate of response rather than byarbitrary numbers of days.

The compound of formula (I) and pharmaceutically acceptable derivativesthereof may be prepared by the general methods outlined hereinafter,said methods constituting a further aspect of the invention.

A comprehensive discussion of the ways in which sensitive groups may beprotected and methods for cleaving the resulting protected derivativesis given by for example T. W. Greene and P. G. M Wuts in ProtectiveGroups in Organic Synthesis 2^(nd) ed., John Wiley & Son, Inc 1991 andby P. J. Kocienski in Protecting Groups, Georg Thieme Verlag 1994 whichare incorporated herein by reference. Examples of suitable aminoprotecting groups include acyl type protecting groups (e.g. formyl,trifluoroacetyl and acetyl), aromatic urethane type protecting groups(e.g. benzyloxycarbonyl (Cbz) and substituted Cbz, and9-fluorenylmethoxycarbonyl (Fmoc)), aliphatic urethane protecting groups(e.g. t-butyloxycarbonyl (Boc), isopropyloxycarbonyl andcyclohexyloxycarbonyl) and benzyl type protecting groups (e.g. benzyl,trityl and chlorotrityl). Examples of suitable oxygen protecting groupsmay include for example silyl groups, such as trimethylsilyl,triethylsilyl or tert-butyldimethylsilyl; ethers such astetrahydropyranyl or tert-butyl; or esters such as acetate or benzoate.Hydroxy groups may be protected by reaction of for example aceticanhydride, benzoic anhydride or a trialkylsilyl chloride in an aproticsolvent. Examples of aprotic solvents are dichloromethane,N,N-dimethylformamide, dimethylsulfoxide, tetrahydrofuran and the like.A carboxylate group may be protected as an ester for example an ethyl orbenzyl ester.

The compounds of general formula (I) and derivatives thereof may bepurified by conventional methods known in the art. For example, thecompounds may be purified by HPLC using an aqueous solution of an acidsuch as formic acid or trifluoroacetic acid with an organic co-solventsuch as acetonitrile or methanol. Alternatively, said compounds may bepurified by crystallisation, chromatography and/or differentialpartition between aqueous and organic solvents.

Compounds of formula (I) may be prepared by reaction of a 4″ aldehydecompound of formula (II)

or a derivative thereof wherein:one or more functional groups may be protected (for example withbenzoyl, triethyl silyl, benzyloxycarbonyl or by formation of a bicyclicketal by interaction of the 9-ketone with the 12-OH and an alcohol, forexample methanol). Alternatively or in addition, the 3′-dimethylaminogroup may be in the form of an N-Oxide or as part of a2′-O-3′-N-bis-benzyloxycarbonyl moiety as described by Flynn et al (J.Amer. Chem. Soc. 1955, 77, 3104-3106),with the amine of formula (III), wherein R¹ is hydrogen or a carboxylicacid protecting group, and R* represents hydrogen or methyl, in areductive amination reaction,

which may be carried out in a suitable solvent, such as DCM, methanol,dioxan or DMF, under neutral to mildly acidic conditions. Suitablereducing agents include, for example, sodium cyanoborohydride, sodiumtriacetoxyborohydride, tetrabutylammonium triacetoxyborohydride or apolymer bound borohydride. A borohydride in a solvent containing aceticacid wherein the triacetoxyborohydride is formed in situ may also beemployed. Alternatively, palladium on charcoal and hydrogen may beemployed to effect the reduction. Suitable reagents for adjustingacidity include acetic acid and sodium acetate.

Appropriate chemical transformations are then carried out to achieve,where necessary and in any order, methylation of the nitrogen in thelinker, re-methylation of the 3′ amine and removal of one or moreprotecting groups as described above.

Methylation of the nitrogen in the linker and/or re-methylation of the3′ position may be effected preferably by a reductive alkylationreaction with formaldehyde. This may be carried out in a solvent, suchas DCM, methanol or DMF, under neutral to mildly acidic conditions.Suitable reducing agents include, for example, sodium cyanoborohydride,sodium triacetoxyborohydride, tetrabutylammonium triacetoxyborohydride,or a polymer bound borohydride. A borohydride in a solvent containingacetic acid wherein triacetoxyborohydride is formed in situ may also beemployed. Alternatively, palladium on charcoal and hydrogen may beemployed to effect the reduction. Suitable reagents for adjustingacidity include acetic acid and sodium acetate. Suitable temperaturesare in the region 0-50° C. typically 20° C. Alternatively reductivemethylation may be achieved using aqueous formaldehyde and formic acidat elevated temperatures for example in refluxing chloroform.

Methylation may also be effected by treatment with an alkylating agent,for example methyl iodide in the presence of a base, for exampleN(CH₂CH₃)(CH[CH₃]₂)₂ or potassium carbonate.

Compound (II) or a derivative thereof may be prepared from compound(IIa)

or a derivative thereof wherein:one or more functional groups may be protected (for example withbenzoyl, triethyl silyl, benzyloxycarbonyl or by formation of a bicyclicketal by interaction of the 9-ketone with the 12-OH and an alcohol, forexample methanol). Alternatively or in addition, the 3′-dimethylaminogroup may be in the form of an N-Oxide or as part of a2′-O-3′-N-bis-benzyloxycarbonyl moiety as described by Flynn et al (J.Amer. Chem. Soc. 1955, 77, 3104-3106),

by oxidative cleavage, for example, using osmium tetroxide and sodiumperiodate. Alternatively ozone at a low temperature, 0° C. or below,typically −50 to −78° C. in the presence of an acid, for example TFA(trifluoroacetic acid) may be employed. In the absence of acid,oxidation of —N(CH₃)₂ on the moiety below, may occur

The N-oxide may be reduced at various stages of the process, asrequired, for example, by treatment with a suitable reducing agent, suchas triphenyl phosphine under appropriate conditions as described in J.of Antib. 41 (1988) 1029-1047 or with Zn powder as described in EP 0 985679.

A compound of formula (IIa) or a derivative thereof, can be formed bypalladium-catalysed allylation of a suitably protected 4″ hydroxycompound, for example by 2′,11-bis-silylation and conversion of the9-ketone to a bicyclic ketal by interaction with the 12-OH and analcohol, for example methanol. Further guidance to O-allylation can begained from Stoner et al J. Org Chem 68, 8847-8852, references therein(palladium catalysed allylation of 6-OH in erythromycin derivatives) andFreiberg et al in U.S. Pat. No. 5,288,709 (4″-O allylation usingNaN(TMS)₂/allyl bromide on a highly modified and protected erythromycinderivative).

In another embodiment of the invention compounds of formula (I), may beprepared by reaction of a compound of formula (IIb) wherein L is asuitable leaving group such as halogen, mesylate or tosylate,

or a derivative thereof wherein:one or more functional groups may be protected (for example withbenzoyl, triethyl silyl, benzyloxycarbonyl or by formation of a bicyclicketal by interaction of the 9-ketone with the 12-OH and an alcohol, forexample methanol). Alternatively or in addition, the 3′-dimethylaminogroup may be in the form of an N-Oxide or as part of a2′-O-3′-N-bis-benzyloxycarbonyl moiety as described by Flynn et al (J.Amer. Chem. Soc. 1955, 77, 3104-3106),with a compound of formula (III) wherein:R¹ is hydrogen or an carboxylic acid protecting group, and R* representshydrogen, methyl or a nitrogen protecting group which facilitatesdeprotonation of the NH group, such as benzyloxycarbonyl,in an appropriate aprotic solvent, such as DCM, DMF or THF, at atemperature of −78 to 100° C. such as 0 to 50° C., in the presence of abase, for example LiN(TMS)2, DBU, potassium carbonate or a stericallyhindered tertiary amine, for example N(CH₂CH₃)(CH[CH₃]₂)₂,

Appropriate chemical transformations are then carried out to achieve,where necessary and in any order, methylation of the nitrogen in thelinker, re-methylation of the 3′ amine and removal of one or moreprotecting groups as described above.

In another embodiment of the invention, compounds of formula (I) may beprepared by reaction of a compound of formula (IV):

or a derivative thereof; wherein:R* represents methyl, hydrogen or a nitrogen protecting group whichfacilitates deprotonation of the NH group, such as benzyloxycarbonyl;and one or more functional groups may be protected (for example withbenzoyl, triethyl silyl, benzyloxycarbonyl or by formation of a bicyclicketal by interaction of the 9-ketone with the 12-OH and an alcohol, forexample methanol). Alternatively or in addition, the 3′-dimethylaminogroup may be in the form of an N-Oxide or as part of a2′-O-3′-N-bis-benzyloxycarbonyl moiety as described by Flynn et al (J.Amer. Chem. Soc. 1955, 77, 3104-3106),with the alkylating agent (V), wherein R¹ is hydrogen or a carboxylicacid protecting group and L is a suitable leaving group such as halogen,mesylate or tosylate,

in an appropriate aprotic solvent such as DCM, DMF or THF at atemperature of −78 to 100° C. such as 0 to 50° C., in the presence of abase for example LiN(TMS)2, DBU, potassium carbonate or a stericallyhindered tertiary amine, for example N(CH₂CH₃)(CH[CH₃]₂)₂.

Appropriate chemical transformations are then carried out to achieve,where necessary and in any order, methylation of the nitrogen in thelinker, re-methylation of the 3′ amine and removal of one or moreprotecting groups as described above.

In another embodiment of the invention, compounds of formula (I) may beprepared by reacting a compound of formula (IV) or a derivative thereof;wherein R* represents methyl or hydrogen, wherein one or more functionalgroups may be protected (for example with benzoyl, triethyl silyl,benzyloxycarbonyl or by formation of a bicyclic ketal by interaction ofthe 9-ketone with the 12-OH and an alcohol, for example methanol).Alternatively or in addition, the 3′-dimethylamino group may be in theform of an N-Oxide or as part of a 2′-0-3′-N-bis-benzyloxycarbonylmoiety as described by Flynn et al (J. Amer. Chem. Soc. 1955, 77,3104-3106),

with a compound of formula (Va)

wherein R¹ is hydrogen or a carboxylic acid protecting group,in a reductive alkylation reaction which may be carried out in asuitable solvent, such as DCM, methanol or DMF, under neutral to mildlyacidic conditions. Suitable reducing agents include, for example, sodiumcyanoborohydride, sodium triacetoxyborohydride, tetrabutylammoniumtriacetoxyborohydride or a similar polymer bound borohydride. Sodiumborohydride in a solvent, such as acetic acid wherein thetriacetoxyborohydride is formed in situ may also be employed.Alternatively, palladium on charcoal and hydrogen may be employed toeffect the reduction. Suitable reagents for adjusting acidity includeacetic acid and sodium acetate.

Appropriate chemical transformations are then carried out to achieve,where necessary and in any order, methylation of the nitrogen in thelinker, re-methylation of the 3′ amine and removal of one or moreprotecting groups as described above.

Compound (IIb) or a derivative thereof can be prepared by converting theprimary alcohol in compound (IVa) or a derivative of it:

into a suitable leaving group such as tosyl, mesyl or halogen byreactions well known to the skilled person.

Compound (IVa) or a derivative thereof may be prepared by treatment ofcompound (II) or a derivative thereof with a reducing agent such assodium borohydride.

All the chemistry above may also be carried out with analogues of (III),(V) or (Va) containing a double or triple bond as shown below:

If intermediates (III′), (III″), (V′), (V″), (Va′) or (Va″) are utilisedthen an additional hydrogenation step is carried out to reduce theunsaturated bond before the compound of formula (I) is obtained.

Compounds of formula (III), (III′) and (III″), may be prepared as shownin scheme 1 below.

In the above scheme:

P¹ represents an amine protecting group, such as Boc orbenzyloxycarbonyl, and R* is hydrogen or methyl, and the dotted bond isa single, double or triple bond.

In step 1) the acid is converted to an acid chloride using a reagentsuch as phosgene, oxallyl chloride or thionyl chloride with DMF ascatalyst at a temperature of 0-40° C., typically 20° C. for about 3 h.Alternative starting materials may have, for example, chlorine, bromineor a sulfonyloxy group, for example triflate, in place of the iodineatom, and chlorine or a sulfonyloxy group for example triflate in placeof the fluorine atom.

In step 2) reaction with ethyl 3-(dimethylamino)acrylate may be effectedin DMF or another suitable solvent at a temperature of 60-120° C.typically 90° C. for about 2.5 h.

In step 3) reaction with N,N-dimethyl hydrazine occurs at temperaturesbetween 0-40° C., typically 20° C. for about 1.5 h.

In step 4) cyclisation takes place in DMF or another suitable solvent inthe presence of a base such as potassium carbonate at a temperature of50-120° C. typically 70° C. for about 2 h.

In step 5) a Sonogashira reaction of a suitable acetylene occurs in thepresence of a suitable catalyst for example copper (I) iodide anddichlorobis(triphenylphosphine)palladium (IIb) in a suitable solvent forexample acetonitrile and base for example triethylamine at temperaturesbetween 0-80° C., typically 20° C. for about 1.5 h.

Where P¹=benzyloxycarbonyl and R*=H, a compound of type (III″) whereR*=benzyloxycarbonyl is thereby obtained.

In step 6) hydrogenation of the unsaturated bond takes place in asuitable solvent for example DCM, ethanol or a mixture of the two, usinga catalyst, for example palladium on carbon, typically at 20° C. with atypical reaction time of 24 h. In the case where P¹ is benzyloxycarbonylthis group will also be removed to give an intermediate (III).

In step 7) where P¹ is Boc, treatment with a strong acid, for exampleHCl in dioxan or TFA in DCM, removes the Boc protection.

In step 8) treatment with a base such as potassium carbonate in a twophase system such as water/DCM or chloroform at temperatures between0-30° C., typically 20° C. gives the amino ester free base.

In step 9) a Heck reaction using a suitable olefin may be carried out togive an intermediate where the dotted bond is a double bond.Alternatively, a Suzuki reaction with a borane, derived for example byhydroboration of a suitable olefin, may be carried out to give a productwhere the dotted bond is a single bond. In these cases, whereP¹=benzyloxycarbonyl and R*=H, a compound of type (III) or (III′) whereR*=benzyloxycarbonyl will be obtained.

Such an intermediate where the dotted line shown represents either asingle or double bond and where P¹ is Boc may be subjected to theconditions of step 7 and step 8 to give a compound of type (III) or(III′).

Such an intermediate where the dotted line shown represents a doublebond and where P¹ is Boc may be subjected to the conditions of steps 6,7 and 8 to give a compound of type (III).

Compound (IV) or a derivative thereof may be prepared by reductiveamination of compound (II) or a derivative thereof, for example usingthe general conditions described above for reductive aminations.

Compounds of formula (V) and (Va), defined above, can be prepared byemploying the method shown in scheme 2 below.

In step 1) a Sonogashira reaction with propargyl alcohol can be effectedin the presence of a suitable catalyst for example copper (I) iodide anddichlorobis(triphenylphosphine)palladium (II) in a suitable solvent, forexample acetonitrile, and base, for example triethylamine, attemperatures between 0-80° C., typically 20° C. for about 1 h.Alternatively, a Heck reaction using allyl alcohol may be carried out togive an intermediate where the dotted bond is a double bond.Alternatively, a Suzuki reaction with a borane, derived for example byhydroboration of allyl alcohol, may be carried out to give a productwhere the dotted bond is a single bond. Alternative starting materialsmay have chlorine, bromine or a sulfonyloxy group, for example triflate,in place of the iodine atom.

Where required, step 2) may be carried out in which hydrogenation of amultiple bond takes place in a suitable solvent for example DCM, ethanolor a mixture of the two, using a catalyst, for example palladium oncarbon, typically at 20° C.

In step 3) oxidation of the alcohol to an aldehyde is carried out, forexample, using Swern conditions (DMSO and oxalyl chloride in thepresence of triethylamine) or manganese dioxide to give aldehydes oftype (Va), (Va′) or (Va″) where the dotted line represents a single,double or triple bond respectively.

In step 4) a leaving group, such as halogen, mesylate or tosylate isintroduced by chemistry well known to those skilled in the art to givecompounds of formula (V), (V′) or (V″) where the dotted line representsa single, double or triple bond respectively.

A compound of formula (I) may also be prepared by coupling a compound offormula (X) or a derivative thereof; wherein:

the dotted line represents a double or triple bond, R* representsmethyl, hydrogen or a nitrogen protecting group such asbenzyloxycarbonyl; and one or more functional groups may be protected(for example with benzoyl, triethyl silyl, benzyloxycarbonyl or byformation of a bicyclic ketal by interaction of the 9-ketone with the12-OH and an alcohol, for example methanol). Alternatively or inaddition, the 3′-dimethylamino group may be in the form of an N-Oxide oras part of a 2′-O-3′-N-bis-benzyloxycarbonyl moiety as described byFlynn et al (J. Amer. Chem. Soc. 1955, 77, 3104-3106) and where thedotted line represents a double or triple bond,with a suitable 6-halo quinolone in a Sonogashira, Heck or Suzukireaction.

Appropriate chemical transformations are then carried out to achieve,where necessary and in any order, methylation of the nitrogen in thelinker, re-methylation of the 3′ amine, reduction of an unsaturated bondin the linker and removal of one or more protecting groups as describedabove.

A compound of formula (X), or a derivative thereof may be prepared byreacting a compound of type (II) or (lib) or a derivative of either witha suitable reagent for example N-methylpropargylamine in an alkylationor reductive alkylation reaction. An intermediate of formula (X) mayalso be prepared by reacting a compound of type (IV) or a derivativethereof with a suitable reagent for example propargyl bromide orpropenal in an alkylation or reductive alkylation reaction.

In another embodiment of the invention, compounds of formula (I) may beprepared by reacting a compound (VI)

or a derivative thereof wherein:one or more functional groups other than the 4″-hydroxyl may beprotected (for example with benzoyl, triethyl silyl, benzyloxycarbonylor by formation of a bicyclic ketal by interaction of the 9-ketone withthe 12-OH and an alcohol, for example methanol). The ketone could alsobe protected, as for example, an oxime. Alternatively or in addition,the 3′-dimethylamino group may be in the form of an N-Oxide or as partof a 2′-O-3′-N-bis-benzyloxycarbonyl moiety as described by Flynn et al(J. Amer. Chem. Soc. 1955, 77, 3104-3106), or the 9-ketone may beprotected as an oxime derivative,with a compound of formula (VII)

wherein R¹ is hydrogen or a carboxylic acid protecting group, and R*represents hydrogen methyl or a nitrogen protecting group such asbenzyloxycarbonyl, and L is a suitable leaving group such as halogen,mesylate or tosylate, and X is oxygen or the C═X moiety is CH₂;in an appropriate aprotic solvent, such as DCM, DMF, DMSO or THF, at atemperature of −78 to 100° C., such as 0 to 50° C., in the presence of abase, for example LiN(TMS)₂, DBU, potassium tert-butoxide, potassiumcarbonate or a sterically hindered tertiary amine, for exampleN(CH₂CH₃)(CH[CH₃]₂)₂.

Appropriate chemical transformations are then carried out to achieve,where necessary and in any order, methylation of the nitrogen in thelinker, re-methylation of the 3′ amine, and removal of one or moreprotecting groups as described above. Where a compound of formula (VII)in which X is oxygen is utilised, reduction of the amide to an amine isalso required.

A compound of formula (VII), as defined above, where the C═X moiety isCH₂, can be prepared by reacting a compound of formula (III) withethylene oxide or a haloethanol in the presence of a suitable base whereappropriate in a suitable solvent for example ethanol, DCM or DMFfollowed by conversion of the resulting hydroxyl group to a leavinggroup by reaction with for example thionyl chloride or a sulfonicanhydride in the presence of a suitable base where appropriate in asuitable solvent for example DCM or THF.

A compound of formula (VII), as defined above, where X is oxygen, can beprepared by reacting a compound of formula (III) with a halo acetylderivative for example chloroacetyl chloride in the presence of asuitable base for example pyridine in a suitable solvent for exampledichloromethane.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

In order that the invention may be more fully understood the followingexamples are given by way of illustration only.

The following abbreviations may appear in the text: 9-BBN for9-borabicyclo[3.3.1]nonane, BOC for t-butoxycarbonyl, DBU for1,8-diazabicyclo[5.4.0]undec-7-ene, DCM for dichloromethane, DMAP for4-dimethylaminopyridine, DMF for N,N-dimethylformamide, DMSO fordimethyl sulfoxide, EtOAc for ethyl acetate, EtOH for ethanol, MeCN foracetonitrile, MeOH for methanol, TFA for trifluoroacetic acid, THF fortetrahydrofuran, MgSO₄ for anhydrous magnesium sulphate and Na₂SO₄ foranhydrous sodium sulphate.

In the procedures that follow, reference to an Intermediate by number istypically provided. This is provided merely for assistance to theskilled chemist to identify the starting material used. The startingmaterial may not necessarily have been prepared from the batch referredto. In addition, the preparation of an Example compound is typicallypresented as a series of individual reaction steps, for example (a),(b), (c), etc. This is also provided merely for assistance to theskilled chemist to identify a suitable sequence of reaction steps toprepare the Example. Although each of the reaction steps indicated willhave been carried out as described, the steps (a), (b), (c), etc. maynot have been performed in one continuous sequence from the same batchof starting materials.

EXAMPLES

Where Example compounds are isolated as salts these are typicallycharacterised and the stoichiometry determined using proton NMR, forexample by considering the chemical shift values, the integrated numberof protons, and by assignment of one or more equivalent peak(s) from theacid and from the parent base.

Intermediate 1 4″-O-Allyl-6-O-methyl-erythromycin A

a) 4″-O-(1-Imidazol-1-yl-carbonyl)-6-O-methyl-erythromycin A

6-O-Methyl-erythromcyin A (108 g) in THF (500 mL) under an atmosphere ofargon was treated portionwise with carbonyldiimidazole (43.2 g) with icebath cooling, the cooling bath was then removed. After 24 h additionalTHF (300 mL) and water (20 mL) were added followed by the dropwiseaddition of water (500 mL). After stirring for 3 h a thick whiteprecipitate had formed. The solid was filtered off, washed with coldwater and dried under vacuum to yield the title compound (107.58 g) as awhite solid. The mother liquors yielded further title compound (9.0 g);ESMS m/z 842.7 [M+H]⁺.

b) 4″-O-(Allyloxycarbonyl)-6-O-methyl-erythromycin A

4″-O-(1-Imidazol-1-yl-carbonyl)-6-O-methyl-erythromycin (50 g) in DCM(200 mL) was cooled to 0° C. under argon and treated with allyl alcohol(23.8 mL) and DBU (9.1 mL). The reaction was stirred at 0° C. for 2 hand at 20° C. for 2 h. The reaction mixture was quenched with 3% aqueouscitric acid (200 mL), the phases separated, and the organic phase washedwith saturated sodium hydrogen carbonate. After drying and evaporationto dryness, the residue was triturated with petroleum ether (bp 40-60°C.) with stirring then filtered and dried to give the title compound asa white solid (41 g); ESMS m/z 832.6 [M+H]⁺.

c)(9S)-4″-O-(Allyloxycarbonyl)-9-dihydro-9-methoxy-2′,11-bis-O-trimethylsilyl-9,12-anhydro-6-O-methyl-erythromycinA mono acetonitrile solvate

4″-O-(Allyloxycarbonyl)-6-O-methyl-erythromycin A (10 g) dissolved indry pyridine (30 mL) was treated with chlorotrimethylsilane (12.0 mL).After stirring for 1 h the pyridine was evaporated and the residuepartitioned between toluene/diethyl ether and saturated aqueous sodiumhydrogen carbonate. The organic layer was separated and the aqueousphase extracted with a fresh portion of diethyl ether. The combinedorganic layers were washed with brine, dried and evaporated to yield thetitle compound as a foam (10.5 g); ESMS m/z 990.8 [M+H]⁺.

This foam was dissolved in acetonitrile (˜100 mL) and the resultantsolution cooled to about 5° C. A seed crystal (prepared by purifying aprevious sample of the title compound by chromatography on silica geleluting with 33-70% diethyl ether in hexane followed by maintaining a100 mg/mL solution in acetonitrile at approx 5° C. for two weeks) wasadded and immediate crystallisation commenced. After standing at about5° C. overnight the mixture was filtered to yield the title compoundmono acetonitrile solvate as a crystalline solid (5.2 g).

d) 4″-O-Allyl-6-O-methyl-erythromycin A

(9S)-4″-O-(Allyloxycarbonyl)-9-dihydro-9-methoxy-2′,11-bis-O-trimethylsilyl-9,12-anhydro-6-O-methyl-erythromycinA mono acetonitrile solvate (15.0 g) in dry THF (100 mL) under argon wastreated with tetrakis(triphenylphosphine)palladium (0.36 g) and theresultant mixture heated at reflux for 1.5 h. Allyl t-butyl carbonate (5mL) (F. Houlihan et al, Can. J. Chem. 1985, 63, 153) was added andheating continued for a further 3.75 h. After cooling and standingovernight at 20° C. the THF was evaporated and the dark brown residuetaken up in 40/60 petroleum ether (100 mL). The solution was treatedwith charcoal, filtered and evaporated. The solid was then taken up inacetonitrile, re-evaporated and dried under vacuum overnight to yield15.89 g. The product (12.8 g) was dissolved in acetonitrile (25 mL) and10% aqueous acetic acid (130 mL). After stirring at 20° C. for 6 hdiethyl ether (50 mL) was added and the layers separated, the organiclayer was extracted with water and the combined aqueous extracts madebasic by the addition of potassium carbonate. The organic product wasextracted with EtOAc (2×100 mL), dried and evaporated to give the titlecompound as a solid (10.0 g); ESMS m/z 788.7 [M+H]⁺.

e) 4″-O-Allyl-6-O-methyl-erythromycin A

4″-O-(Allyloxycarbonyl)-6-O-methyl-erythromycin A (272.02 g) wasdissolved in DCM (1360 mL) and DBU (208 mL) was added. The solution wascooled to 0-5° C. and triethylsilyl chloride (220 mL.) was addeddropwise over 50 minutes. The reaction mixture was allowed to warm up toroom temperature and stirred for 24 h.

The reaction mixture was washed successively with water (0.98 L),saturated NaHCO₃ (0.98 L) and the aqueous phase extracted with DCM. Thecombined organic phases were washed with brine (0.98 L). The organicphase was concentrated under reduced pressure. The residue was dissolvedin methanol (1 L) and pyridine hydrochloride (37.79 g) in methanol (360mL) added. The reaction mixture was stirred for 18 h. DCM (2.72 L) wasadded and the solution was washed with water (1.632 L), and saturatedNaHCO₃ (1.632 L). The organic phase was evaporated under reducedpressure. The residue was dissolved in THF (1.65 L) and evaporated underreduced pressure. This was repeated. The residue was dissolved in THF(3300 mL), degassed and flushed with nitrogen. Pd(PPh₃)₄ (14.82 g) wasadded and the solution heated to reflux and stirred under nitrogen for 6hours.

More Pd(PPh₃)₄ (1.50 g) in THF (120 mL) was added over 10 min followedby dropwise addition of tertbutylallylcarbonate (162 mL) over 30 min.The reaction mixture was stirred at 65° C. for 16 hours. The solutionwas cooled to 60° C., activated charcoal (27.5 g) added and the mixtureheated to reflux for 40 min, then cooled to room temperature, filteredthrough celite and the filtered solid washed with THF (2 L).

The filtrate was concentrated under vacuum, the residue was dissolved inacetonitrile (1 L) and evaporated under reduced pressure. This wasrepeated. The residue was dissolved in acetonitrile (1 L) and aceticacid (3 L) and water (5 L) added. The reaction mixture was stirred atroom temperature for 70 hours.

The reaction mixture was basified to pH 9 with solid K₂CO₃. Theresulting suspension was extracted twice with EtOAc (2×2.5 L). Thecombined organic phases were washed with water (2 L) and concentrated by80% under vacuum. Methylcyclohexane (1 L) was added and the mixtureconcentrated by 80% under vacuum. This was repeated, then a thirdportion of methylcyclohexane (1 L) was added and the suspension filteredoff. The solid was washed with methylcyclohexane (3×350 mL) and driedunder vacuum at 42° C. to give the title compound as a white powder(115.4 g). δ_(c) (100 MHz; CDCl₃) 9.0, 10.5, 12.3, 15.9, 16.0, 18.0,19.0, 19.7, 20.9, 21.5, 21.6, 28.4, 35.5, 37.1, 39.0, 39.2, 40.1, 44.9,45.3, 49.6, 50.6, 64.7, 64.8, 68.0, 69.0, 71.0, 73.4, 74.2, 75.0, 76.5,78.2, 78.4, 80.2, 86.5, 96.2, 102.3, 117.1, 134.8, 175.9, 221.1.

Intermediate 2 4″-O-(2-oxoethyl)-6-O-methyl-erythromycin A

4″-O-Allyl-6-O-methyl-erythromycin A (95.8 g) in DCM (1 L) and methanol(100 mL) was cooled to −70° C. under argon and TFA (18.8 mL) added.Ozonized oxygen was bubbled through until a blue colour developed (1.25h). Argon was bubbled through the mixture to flush out the ozone, thendimethyl sulfide (35.7 mL) and triethylamine (50.5 mL) were added. Thereaction was stirred at −70° C. for 30 min then removed from the coolingbath. After 0.5 h the reaction (now at −30° C.) was warmed to 0° C. in awater bath and stirred for a further 0.5 h.

The reaction mixture was washed with water (500 mL), dried (Na₂SO₄) andevaporated to dryness. The residue was dissolved in toluene andevaporated under reduced pressure (three times each 300 mL) to give thetitle compound (103.7 g) which was used without purification; ESMS m/z822.7 [M+MeOH+H]⁺, 834.6 [M+HCO₂]⁻.

Intermediate 3 Ethyl6-(3-methylaminopropyl)-1-(dimethylamino)-4-oxo-1,4-dihydro-3-quinolinecarboxylate

a) Ethyl 3-(dimethylamino)-2-(2-fluoro-5-iodobenzoyl)-2-propenoate

A stirred suspension of 2-fluoro-5-iodobenzoic acid (99.7 g) in DCM (1L) at 20° C. was treated with oxalyl chloride (49.8 mL) and DMF (0.3mL). After 3 h further DMF (0.1 mL) was added. After a further 2 h theclear solution was evaporated and re-evaporated from toluene (3×200 mL).The acid chloride was re-dissolved in toluene (1.5 L) and treated withtriethylamine (79.2 mL) and ethyl 3-(dimethylamino)acrylate (65.3 g).After stirring for 2.5 h at 90° C. the mixture was filtered andevaporated. The residue was redissolved in EtOAc, washed with saturatedsodium hydrogen carbonate solution (2×), water, saturated brine, dried(MgSO₄) and treated with decolourising charcoal for 0.5 h. The mixturewas filtered, evaporated, redissolved in diethyl ether and allowed tocrystallise. The solid was filtered off, washed with diethyl ether anddried to give the title compound (91.2 g). On concentration and seedinga second crop was obtained (9.0 g); ESMS m/z 391.9 [M+H]⁺.

b) Ethyl1-(dimethylamino)-6-iodo-4-oxo-1,4-dihydro-3-quinolinecarboxylate

A stirred suspension of Intermediate 3a (50 g) in EtOH (500 mL) wastreated with 1,1-dimethylhydrazine (10.7 mL). After stirring for 2.5 hthe clear solution was evaporated. The residue was dissolved in DMF (500mL), treated with potassium carbonate (26.5 g) and the mixture stirredat 70° C. for 2 h. After cooling to 20° C. the mixture was poured intowater containing excess citric acid, the solid filtered off, washed withwater and dried to give the title compound (48.07 g); ESMS m/z 386.9[M+H]⁺.

c) Ethyl6-(3-t-butoxycarbonylmethylaminopropyn-1-yl)-1-(dimethylamino)-4-oxo-1,4-dihydro-3-quinolinecarboxylate

A stirred mixture of Intermediate 3b (48.07 g) and copper (I) iodide(2.36 g) in triethylamine (510 mL) and MeCN (1 L) was stirred underargon for 15 min. N-t-butoxycarbonyl-N-methylpropargylamine (31.72 g)and dichlorobis(triphenylphosphine)palladium (II) (2.8 g) were added.After 1.5 h the mixture was evaporated and redissolved in DCM. Thesolution was washed with saturated sodium hydrogen carbonate solution(2×), water, saturated brine, dried (Na₂SO₄) and evaporated. The residuewas triturated with diethyl ether. The solid was filtered off washedwith diethyl ether and dried, then boiled in EtOAc, filtered, thesolution diluted with diethyl ether and allowed to crystallise to givethe title compound (13.88 g). The solid insoluble in hot EtOAc wasdissolved in hot ethanol and treated with decolourising charcoal. Thehot mixture was filtered and evaporated to give more of the titlecompound (32.57 g); δ_(H) (250 MHz; CDCl₃) 1.43 (3H, t, J=7.1 Hz), 1.50(9H, s), 2.92 (6H, s), 2.99 (3H, s), 4.29 (2H, br s), 4.42 (2H, q, J=7.1Hz), 7.68 (1H, dd, J=1.9 & 8.8 Hz), 8.02 (1H, d, J=8.8 Hz), 8.50 (1H, d,J=1.9 Hz), 8.73 (1H, s).

d) Ethyl6-(3-t-butoxycarbonylmethylaminopropyl)-1-(dimethylamino)-4-oxo-1,4-dihydro-3-quinolinecarboxylate

A solution of Intermediate 3c (32.57 g) in DCM (300 mL) was treated with10% Pd/C (2 g) and stirred for 1 min. The catalyst was filtered off andreplaced with fresh (2 g). The mixture was hydrogenated at ambienttemperature and pressures overnight, filtered and evaporated. Theresidue was triturated with diethyl ether. The solid was filtered offwashed with diethyl ether and dried to give the title compound (26.33g). The ether solution was evaporated, redissolved in EtOH and treatedwith decolourising charcoal. Filtration, evaporation and triturationwith diethyl ether gave a second crop (3.96 g); ESMS m/z 432.2 [M+H]⁺.

e) Ethyl6-(3-methylaminopropyl)-1-(dimethylamino)-4-oxo-1,4-dihydro-3-quinolinecarboxylate

A stirred solution of Intermediate 3d (30.29 g) in DCM (120 mL) wastreated with 4M HCl in 1,4-dioxan (120 mL). After 1 h the mixture wasevaporated and the residue partitioned between DCM and 10% K₂CO₃solution. The DCM solution was collected, washed with saturated brine,dried (Na₂SO₄) and evaporated. The gummy solid was triturated withdiethyl ether then light petroleum 40-60 was added portionwise. Thesolid was filtered off, washed with light petroleum 40-60 and dried toyield the title compound (19 g); ESMS m/z 332.1 [M+H]⁺.

f) Ethyl6-(3-methylaminopropyl)-1-(dimethylamino)-4-oxo-1,4-dihydro-3-quinolinecarboxylate

To a stirred solution of ethyl6-(3-t-butoxycarbonylmethylaminopropyl)-1-(dimethylamino)-4-oxo-1,4-dihydro-3-quinolinecarboxylate(518 g) in chloroform (2070 mL) was added a mixture of 4M HCl in dioxane(1070 mL, containing 20-30% water) over 1 hour. The reaction was stirredfor a further 1 h. The phases were separated, the organic layer wasconcentrated in vacuo and combined with the aqueous layer. To this wasadded chloroform (3 L). Potassium carbonate (475 g) in water (4 L) wasadded, the organics were separated and the aqueous re-extracted withchloroform (3 L and 1.5 L). The combined organics were washed with brine(3 L) before being dried over magnesium sulphate, filtered andconcentrated in vacuo to give a solid. The material was triturated withdiethyl ether (1.5 L) and stirred for 20 mins before the addition ofhexane (750 L). The suspension was stirred for a further 10 mins beforebeing filtered and dried to give 328 g, of the title product as an offwhite solid.

DIMS m/z 332.2 [M+H]⁺.

Example 14″-O-{2-[(3-[3-Carboxy-1-dimethylamino-4-oxo-1,4-dihydro-6-quinolinyl]propyl)methylamino]ethyl}-6-O-methyl-erythromycinA D-tartrate salt

a)4″-O-{2-[(3-[3-Carboxy-1-dimethylamino-4-oxo-1,4-dihydro-6-quinolinyl]propyl)methylamino]ethyl}-6-O-methyl-erythromycinA ethyl ester Intermediate 2 (91.5 g) in DCM (1 L) was stirred underargon with ethyl6-(3-methylaminopropyl)-1-(dimethylamino)-4-oxo-1,4-dihydro-3-quinolinecarboxylate(33.6 g) and 3 A molecular sieves (45 g) for 30 min, then sodiumtriacetoxy borohydride (45.1 g) was added. After 40 min the solution wasdecanted from the molecular sieves, which were extracted with more DCM.The combined DCM solutions were washed twice with water and once with 5%potassium carbonate, dried (Na₂SO₄) and evaporated under reducedpressure to give the crude product (110.6 g). This was purified using 2Biotage 75 800 g columns eluting with 0-6.4% (10:1 MeOH/0.880 NH₃) inDCM to give the title compound (65.1 g); ESMS m/z 1106.0 [M+H]⁺.

b)4″-O-{2-[(3-[3-Carboxy-1-dimethylamino-4-oxo-1,4-dihydro-6-quinolinyl]propyl)methylamino]ethyl}-6-O-methyl-erythromycinA D-tartrate salt Example 1a (65.1 g) in THF (550 mL) was treated with0.5M LiOH (283 mL) at room temperature under argon. After 2.5 h 1 Msodium dihydrogen phosphate was added to give a pH of approximately 11,and the mixture extracted with diethyl ether (500 mL). The aqueous layerwas treated with a small amount of phosphoric acid, giving a pH of 10.4when a solid rapidly separated. This was collected by filtration andwashed with diethyl ether and water to give 125 g of wet solid. Aportion of this (109 g) was slurried in water and ethyl acetate and thepH adjusted to 7.2 with 1 M sodium dihydrogen phosphate. The ethylacetate was evaporated under reduced pressure and the aqueous extractedwith 2×400 mL DCM, the combined extracts were dried (Na₂SO₄) andevaporated under reduced pressure to give the product free base (47.8g). This material was dissolved in acetone (3.2 L) at 45° C. and treatedwith D-tartaric acid (6.66 g in 240 mL water) and seeded with authenticmaterial. After stirring and allowing to cool for 2 h followed by icebath cooling to 15° C., the solid was filtered off, washed with acetoneand dried under reduced pressure to give the title compound as a whitepowder (43.9 g); ESMS m/z 1077.9 [M+H]⁺. δ_(H) (500 MHz; CD3OD+1 dropD2O), inter alia 4.32 (2H, s, tartrate) 2.81 (3H, s, 22″-CH₃), 2.84 (6H,7′-, 8′-CH₃).

δ_(c) (125 MHz, HSQC/HMBC, CD3OD+1 drop D2O) 8.6, 9.7, 11.2, 15.0, 15.7,17.1, 18.0, 19.4, 20.5, 20.5, 20.8, 25.3, 30.3, 31.7, 34.5, 37.9 38.4,38.7, 38.8, 39.6, 44.8, 45.0, 45.0, 48.9, 49.9, 55.6, 55.8, 64.4, 64.8,66.6, 67.3, 69.1, 69.4, 73.5, 74.3, 74.7, 76.8, 78.4, 78.4, 80.6, 87.0,96.2, 101.7, 108.7, 117.6, 124.4, 125.6, 135.5, 139.4, 140.0, 143.3,168.0, 177.2, 176.6, 177.7, 221.5.

Example 24″-O-{2-[(3-[3-Carboxy-1-dimethylamino-4-oxo-1,4-dihydro-6-quinolinyl]propyl)methylamino]ethyl}-6-O-methyl-erythromycinA crystallized from acetone/water

Example 1 (18 g) in DCM (150 mL) and water (10 mL) was shaken withsaturated aqueous sodium hydrogen carbonate solution (30 mL). Theaqueous layer was extracted with DCM (3×50 mL), the four organicextracts combined, dried (Na₂SO₄) and evaporated under reduced pressureto a foam. This material was taken up in acetone and evaporated todryness to give a white solid foam, (14.2 g). Similar material fromanother preparation (0.6 g) was added, and the material was crystallisedtwice from acetone/water (1:1, 11 mug) to give the title compound, afterdrying under vacuum over phosphorus pentoxide (10.748 g); mp 144-145°C., ES m/z 1077.6 [M+H]⁺. δ_(H) (400 MHz, CDCl₃) inter alia 2.25 (6H,7′-, 8′-CH₃), 2.26 (3H, s, 22″-CH₃).

Example 34″-O-{2-[(3-[3-Carboxy-1-dimethylamino-4-oxo-1,4-dihydro-6-quinolinyl]propyl)methylamino]ethyl}-6-O-methyl-erythromycinA crystallised from ethanol

Ethanol (3 mL) was added to4″-O-{2-[(3-[3-Carboxy-1-dimethylamino-4-oxo-1,4-dihydro-6-quinolinyl]propyl)methylamino]ethyl}-6-O-methyl-erythromycinA (809.0 mg). To the solution, seeds of crystalline free-base (from anethanol crystallisation which had been seeded with crystals produced bycrystallisation from acetone/supercritical fluid carbon dioxide) wereadded. The solution was stirred overnight at room temperature which ledto the formation of crystalline white solid. The solid was filtered,washed with ethanol and analysed using FT-IR and DSC. Obtained about503.5 mg of crystalline free-base.

DSC showed a single endotherm maximum at 192.38° C.

Example 44″-O{2-[(3-[3-Carboxy-1-dimethylamino-4-oxo-1,4-dihydro-6-quinolinyl]propyl)methylamino]ethyl}-6-O-methyl-erythromycinA diphosphate salt

4″-O-{2-[(3-[3-Carboxy-1-dimethylamino-4-oxo-1,4-dihydro-6-quinolinyl]propyl)methylamino]ethyl}-6-O-methyl-erythromycinA (9.1 g) in acetonitrile (450 mL) was treated with a solution ofphosphoric acid (1.656 g) in water (40 mL) and acetonitrile (90 mL). Thehazy solution was filtered through kieselguhr and allowed to crystallizeovernight at 20° C. and 3 h at 4° C. The white solid was filtered off,washed with acetonitrile and dried to give the title compound (8.238 g);ESMS m/z 1077.9 [M+H]⁺, OH (400 MHz, CD3OD) inter alia 2.77 (3H, s,22″-CH₃), 2.84 (6H, 7′-, 8′-CH₃)

Example 54″-O-{2-[(3-[3-Carboxy-1-dimethylamino-4-oxo-1,4-dihydro-6-quinolinyl]propyl)methylamino]ethyl}-6-O-methyl-erythromycinA a) 2′-O-Benzoyl-4″-O-allyl-6-O-methyl-erythromycin A

4″-O-Allyl-6-O-methyl-erythromycin A (5.0 g) in dry DCM (50 mL) underargon was treated with triethylamine (1.41 mL). Benzoyl chloride (0.95mL) was then added dropwise. The resultant mixture was stirred for 2 h,then additional benzoyl chloride (0.14 mL) was introduced. Afterstirring for a further 1 h, the mixture was quenched by the addition of1 N NaOH. The layers were separated and the organic phase was washedwith brine, then dried (Na₂SO₄) and concentrated in vacuo.Chromatography on silica eluting with 0-10% (2M NH₃ in MeOH) in DCM gavethe title compound as a white foam (5.6 g); ES m/z 892.5 [M+H]⁺.

b) 2′-O-Benzoyl-4″-O-(2-oxoethyl)-6-O-methyl-erythromycin A

Example 5a (862 mg) in DCM (20 mL) and methanol (2 mL) was cooled to−70° C. and TFA (143 μL) added. Ozonized oxygen was bubbled throughuntil a blue colour developed. Oxygen and then argon were bubbledthrough the mixture to flush out the ozone, then dimethyl sulfide (283μL) and triethylamine (404 μL) were added. The reaction was then removedfrom the cooling bath and allowed to warm to room temperature. Thereaction mixture was then diluted with water, the organic layer removedand then washed three times with water, dried (Na₂SO₄) and evaporated todryness to afford the title compound which was used withoutpurification; ES m/z 912.5 [M+H₂O+H]⁺.

c)2′-O-Benzoyl-4″-O-{2-[(3-[3-carboxy-1-dimethylamino-4-oxo-1,4-dihydro-6-quinolinyl]propyl)methylamino]ethyl}-6-O-methyl-erythromycinA ethyl ester

Example 5b in DCM (12 mL) was stirred under argon with ethyl6-(3-methylaminopropyl)-1-(dimethylamino)-4-oxo-1,4-dihydro-3-quinolinecarboxylate(301 mg) and 3 A molecular sieves (300 mg), then sodium triacetoxyborohydride (411 mg) was added. After 20 min the solution was decantedfrom the molecular sieves. The DCM solution was washed with water andNaHCO₃ solution. The combined aqueous phases were re-extracted with DCM,the combined DCM solutions were then washed with brine and then dried(Na₂SO₄) and concentrated in vacuo. Chromatography on silica elutingwith 0-17% (2M NH₃ in MeOH) in DCM gave the title compound as acolourless foam (648 mg); ES m/z 1209.6 [M+H]⁺.

d)2′-O-Benzoyl-4″-O-{2-[(3-[3-carboxy-1-dimethylamino-4-oxo-1,4-dihydro-6-quinolinyl]propyl)methylamino]ethyl}-6-O-methyl-erythromycinA

Example 5c (200 mg) in THF (12 mL) was treated with 0.5M LiOH (6 mL) atroom temperature under argon. After 3 h 40 drops of AcOH were added andthe mixture was concentrated in vacuo. Chromatography on silica elutingwith 0-20% (9:1 MeOH/0.880 NH₃) in DCM gave the title compound as acolourless foam (200 mg); ES m/z 1181.6 [M+H]⁺.

e)4″-O-{2-[(3-[3-carboxy-1-dimethylamino-4-oxo-1,4-dihydro-6-quinolinyl]propyl)methylamino]ethyl}-6-O-methyl-erythromycinA

Example 5d (200 mg) in MeOH (10 mL) was refluxed for 48 h under argon.The crude product was triturated with hexanes. Chromatography on silicaeluting with 5-16% (9:1 MeOH/0.880 NH₃) in DCM gave the title compoundas a colourless foam (130 mg); ES m/z 1077.6 [M+H]⁺.

Example 64″-O-{2-[(3-[3-Carboxy-1-dimethylamino-4-oxo-1,4-dihydro-6-quinolinyl]propyl)methylamino]ethyl}-6-O-methyl-erythromycinA difumarate salt

Isopropanol (2 mL) was added to4″-O-{2-[(3-[3-Carboxy-1-dimethylamino-4-oxo-1,4-dihydro-6-quinolinyl]propyl)methylamino]ethyl}-6-O-methyl-erythromycinA, (231.7 mg). The solution was stirred for 15 minutes at roomtemperature and then fumaric acid (52.42 mg) was added to the solution.The slurry was heated to 50° C. and left stirring overnight. The slurrywas filtered, washed with i-propanol and dried in a vacuum oven at 50°C. with a slow flow of nitrogen, to give the title compound (112.2 mg).δ_(H) (400 MHz, (CD3)2SO) inter alia 2.26 (3H, s, 22″-CH₃), 2.44 (6H,7′-, 8′-CH₃), 6.55 (4H, s, fumarate) DSC showed a single endothermmaximum at 215.43° C.

Biological Data

Whole-cell antimicrobial activity was determined by broth microdilutionusing the Clinical and Laboratory Standards (CLSI) recommended procedure(Document M7-A6A7, Methods for Dilution Susceptibility Tests forBacteria that Grow Aerobically). Compounds were dissolved in DMSO anddiluted into water to produce a 640 μg/mL stock solution. The stocksolution was further diluted 1:10 into Haemophilus Test Media to producea 64 μg/mL working stock solution. A Microlab AT Plus 2 (Hamilton Co.,Reno, Nev.) was used to prepare serial two-fold dilutions (50 μLaliquots) of the working stock in a 96 well microtitre plate. After thecompounds were diluted, a 50 μL aliquot of the test isolate (˜1×106cfu/ml) was added to each well of the microtitre plate. The final testconcentrations ranged from 0.016-16 μg/mL. Inoculated plates wereincubated at 35° C. in ambient air for 18 to 24 hours. The minimuminhibitory concentration (MIC) was determined as the lowestconcentration of compound that inhibited visible growth. The compoundsin the above examples generally gave MICs less than 1 μg/mL againsterythromycin-sensitive and erythromycin-resistant strains ofStreptococcus pneumoniae and/or Streptococcus pyogenes.

However, it will appreciated by a person skilled in the art thatcompounds of the invention may have different levels of activity againstdifferent strains of the same bacteria.

The MICs of compounds can be determined against 42 strains ofHaemophilus influenzae. The 90th percentile of these values is termedthe MIC90, while the 50th percentile is termed the MIC50. The geometricmean of the 42 values may also be calculated.

In Vivo Model of Respiratory Tract Infection (H.inf 128)

For these studies, CD-1 mice are acclimated to a restricted diet (8grams/day) for 4 days prior to infection. Once on study, mice receivetheir daily ration each morning. An inoculum is obtained by placing 40μl of Haemophilus influenzae H-128 onto Chocolate II agar plates andputting them into the CO₂ incubator for approximately 18 hours. 20colonies are taken from the plates and put into 50 mls Mueller-Hintonbroth with 5% Fildes and placed into CO₂ incubator for approximately 24hours. The conical tube is centrifuged at 4000 rpms for 20 minutes at37° C. The pellet is resuspended in 3 mls of Mueller Hinton broth with5% Fildes. That tube is serially diluted 1:10 into saline to 10-7.10-5-10-7 dilutions (200 μl each) are plated in triplicate forenumeration of the inoculum (done by scoring colony-forming units aftergrowing overnight in CO₂ incubator). From the original conical tube, a1:5 dilution of bacteria:melted nutrient agar is made and placeddirectly into 38-41° C. water bath.

For the infection, mice are anesthetized with isoflurane using ananesthesia machine. The anesthetized mice are then infected with 20μl/mouse of bacterial suspension in molton nutrient agar by directintra-bronchial instillation via intratrachial intubation. Mice aredosed at 1, 7, 24, 31, 48, 55, 72 and 79 hours post-infection with thecorresponding compounds. Mice are sacrificed at 96 hours by CO₂ overdoseand the lungs excised for enumeration of viable bacteria numbers aftergrowing overnight in CO₂ incubator.

Bioavailability/Oral Exposure

The bioavailability/oral exposure of compounds of the present inventionmay be tested using the following protocols in rat or mouse.

Rat Animal Model—Rats are surgically implanted with a Tygon catheter andare allowed a 4 day post-surgical recovery period prior to studyinitiation. Food (PMI 5002 certified diet) and water are providedad-libitum until placed into a commercial auto sampling system (Culex,Bioanalytical Systems, West Lafayette, Ind.). Animals are acclimated tothe caging and tethering system on the automated system for a minimum of24 hours prior to study initiation. During acclimation and throughoutthe duration of the study, animals are given 5 food pellets/day (≈20 g)and free access to water.

Dose Preparation—Oral target doses in the rat are 50 mg/kg at 16 mL/kgand intravenous target doses are 5 mg/kg at 4 mL/kg. The intravenousdose is infused over a 1 hour period. The final dose Solutioncompositions are: 1.0% DMSO; 20% Encapsin (w/v) in water (po), or saline(iv) pH 3.5-4.0. Dose solution aliquots are taken (N=3, 50-μL) by weightand frozen at −80° C. until subsequent LC/MS/MS analysis along withblood/water samples.

Study regimen and sample collection—During the PO dose session, animalsare orally dosed with a stainless steel gavage needle with a standarddose volume of 16 mL/kg. Rats are then allowed a 2 day washout periodand crossed over for a second IV dose session in which animals areinfused for 1 hour (4 mL/kg) into the femoral vein while blood samplesare removed via the femoral artery catheter. When blood samples areremoved from the femoral artery by the Culex instrument, samples areretained in a fraction collector at 33° F. in heparanized glass tubes.The standard timepoints at which blood samples are collected are asfollows:

po: 0, 15, 30, 45, 60, 90, 120, 180, 240, 360, 480, 720, 960 and 1440min. (75 uL per sample)

iv: 0, 20, 40, 60, 65, 75, 90, 120, 180, 240, 360, 480, 960, 1440 and1800 min. (75 uL per sample)

Duplicate blood samples are aliquoted (25 uL) and 25 uL of water isadded by a Tecan® robotic pipetting instrument. The blood/water lysatesare then stored at −80° C. until subsequent quantitation by LC/MS/MS.

PK analysis—Non-compartmental pharmacokinetic analysis is performedusing WinNonlin version 4.1; NCA and an internal DMPK software program,MPKR 1.74.

Mouse Animal Model—Mice are acclimated to a 2 pellets (≈8 g)/day (PMI5002 certified diet) for a minimum of 3 days and water is providedad-libitum. Mice are individually housed in polycarbonate shoeboxes.

Dose Preparation—Oral target doses in the mice are 300 mg/kg at 16mL/kg. The final dose Solution compositions are: 1.0% DMSO; 20% Encapsin(w/v) in water (po), pH 3.5-5.5. Dose solution aliquots are taken(N=2-3, 50-μL) by weight and frozen at −80° C. until subsequent LC/MS/MSanalysis along with blood/water samples.

Study regimen and sample collection—During the PO dose session, animalsare orally dosed with a stainless steel gavage needle with a standarddose volume of 16 mL/kg. Blood samples are removed from the femoralartery via the lateral tail veins into lithium heparinized microfugetubes. The standard timepoints at which blood samples are collected areas follows: po: 20, 40, 60, 90, 120, 180, 240, 360, 480, and 1440 (25 uLper sample). The blood is aliquoted (25 uL) and 25 uL of water is addedimmediately. The blood/water lysates are then stored at −80° C. untilsubsequent quantitation by LC/MS/MS.

PK analysis—Non-compartmental pharmacokinetic analysis is performedusing WinNonlin version 4.1; NCA and an internal DMPK software program,MPKR 1.74.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer or step or group of integers but not to theexclusion of any other integer or step or group of integers or steps.

The application of which this description and claims forms part may beused as a basis for priority in respect of any subsequent application.The claims of such subsequent application may be directed to any featureor combination of features described herein. They may take the form ofproduct, composition, process, or use claims and may include, by way ofexample and without limitation, the following claims:

1. A compound of formula (I)

or a pharmaceutically acceptable derivative thereof.
 2. The compound4″-O-{2-[(3-[3-Carboxy-1-dimethylamino-4-oxo-1,4-dihydro-6-quinolinyl]propyl)methylamino]ethyl}-6-O-methyl-erythromycinA. 3-6. (canceled)
 7. A method for the treatment of the human ornon-human animal body to combat microbial infection comprisingadministration to a body in need of such treatment of an effectiveamount of a compound as claimed in claim 1, or a pharmaceuticallyacceptable derivative thereof.
 8. A pharmaceutical compositioncomprising a compound as claimed in claim 1, or a pharmaceuticallyacceptable derivative thereof, in association with a pharmaceuticallyacceptable excipient, diluent and/or carrier.
 9. A method for thetreatment of the human or non-human animal body to combat microbialinfection comprising administration to a body in need of such treatmentof an effective amount of a compound as claimed in claim
 2. 10. Apharmaceutical composition comprising a compound as claimed in claim 2in association with a pharmaceutically acceptable excipient, diluentand/or carrier.